[Show abstract][Hide abstract] ABSTRACT: In this study, a moderate thermophile Clostridium thermobutyricum is shown to ferment the sugars in sweet sorghum juice treated with invertase and supplemented with tryptone (10gL(-1)) and yeast extract (10gL(-1)) at 50°C to 44gL(-1) butyrate at a calculated highest volumetric productivity of 1.45gL(-1)h(-1) (molar butyrate yield of 0.85 based on sugars fermented). This volumetric productivity is among the highest reported for batch fermentations. Sugars from acid and enzyme-treated sweet sorghum bagasse were also fermented to butyrate by this organism with a molar yield of 0.81 (based on the amount of cellulose and hemicellulose). By combining the results from juice and bagasse, the calculated yield of butyric acid is approximately 90kg per tonne of fresh sweet sorghum stalk. This study demonstrates that C. thermobutyricum can be an effective microbial biocatalyst for production of bio-based butyrate from renewable feedstocks at 50°C.
[Show abstract][Hide abstract] ABSTRACT: A bio-based process is appealing for purification of L-lactic acid, the major enantiomer of polylactic acid syrup, generated by thermochemical processes at the end of life of PLA-based plastics, from its chiral impurity, D-lactic acid, before reuse.
Polylactic acid (PLA), a renewable alternative to petroleum-derived plastics, contains a mixture of L- and D-lactic acid (LA) isomers with the L-isomer dominating (up to 95 %). A novel bio-based process was developed to produce chirally pure L-LA from syrup produced during recycling of PLA-plastics. This process utilizes an engineered Escherichia coli (strain DC1001) containing novel gene deletions (lld, ykg) that eliminated the oxidative metabolism of L-lactate, leaving the membrane-bound D-lactate dehydrogenases to selectively metabolize the D-isomer. Strain DC1001 removed 8.7 g D-lactate l(-1) from a PLA-syrup containing 135 g total lactic acid l(-1) in 24 h. Average rates of removal of D-lactic acid were 0.25 g D-lactate h(-1) (g cell dry weight)(-1) and 0.36 g D-lactate l(-1) h(-1).
Bio-based purification of PLA-syrup utilizing E. coli strain DC1001 is an attractive process step during recycling of PLA-plastics. This selective oxidation process can also be used to remove chiral contamination of L-lactate in medical applications.
[Show abstract][Hide abstract] ABSTRACT: Pretreatments such as dilute acid at elevated temperature are effective for the hydrolysis of pentose polymers in hemicellulose and also increase the access of enzymes to cellulose fibers. However, the fermentation of resulting syrups is hindered by minor reaction products such as furfural from pentose dehydration. To mitigate this problem, four genetic traits have been identified that increase furfural tolerance in ethanol-producing Escherichia coli LY180 (strain W derivative): increased expression of fucO, ucpA, or pntAB and deletion of yqhD. Plasmids and integrated strains were used to characterize epistatic interactions among traits and to identify the most effective combinations. Furfural resistance traits were subsequently integrated into the chromosome of LY180 to construct strain XW129 (LY180 ΔyqhD ackA::PfucO-ucpA) for ethanol. This same combination of traits was also constructed in succinate biocatalysts (Escherichia coli strain C derivatives) and found to increase furfural tolerance. Strains engineered for resistance to furfural were also more resistant to the mixture of inhibitors in hemicellulose hydrolysates, confirming the importance of furfural as an inhibitory component. With resistant biocatalysts, product yields (ethanol and succinate) from hemicellulose syrups were equal to control fermentations in laboratory media without inhibitors. The combination of genetic traits identified for the production of ethanol (strain W derivative) and succinate (strain C derivative) may prove useful for other renewable chemicals from lignocellulosic sugars.
Proceedings of the National Academy of Sciences 03/2013; 110(10):4021-6. DOI:10.1073/pnas.1217958110 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A process was developed for seed culture expansion (3.6 million-fold) using 5% of the hemicellulose hydrolysate from dilute acid pretreatment as the sole organic nutrient and source of sugar. Hydrolysate used for seed growth was neutralized with ammonia and combined with 1.0mM sodium metabisulfite immediately before inoculation. This seed protocol was tested with phosphoric acid pretreated sugarcane and sweet sorghum bagasse using a simplified process with co-fermentation of fiber, pentoses, and hexoses in a single vessel (SScF). A 6h liquefaction (L) step improved mixing prior to inoculation. Fermentations (L+SScF process) were completed in 72h with high yields (>80gal/USton). Ethanol titers for this L+SScF process ranged from 24g/L to 32g/L, and were limited by the bagasse concentration (10% dry matter).
[Show abstract][Hide abstract] ABSTRACT: Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium isolated from sweetgum (Liquidambar styraciflua) wood, is able to efficiently depolymerize, assimilate and metabolize 4-O-methylglucuronoxylan, the predominant structural component of hardwood hemicelluloses. A basis for this capability was first supported by the identification of genes and characterization of encoded enzymes and has been further defined by the sequencing and annotation of the complete genome, which we describe. In addition to genes implicated in the utilization of β-1,4-xylan, genes have also been identified for the utilization of other hemicellulosic polysaccharides. The genome of Paenibacillus sp. JDR-2 contains 7,184,930 bp in a single replicon with 6,288 protein-coding and 122 RNA genes. Uniquely prominent are 874 genes encoding proteins involved in carbohydrate transport and metabolism. The prevalence and organization of these genes support a metabolic potential for bioprocessing of hemicellulose fractions derived from lignocellulosic resources.
[Show abstract][Hide abstract] ABSTRACT: Pyruvate dehydrogenase (PDH) of Escherichia coli is inhibited by NADH. This inhibition is partially reversed by mutational alteration of the dihydrolipoamide dehydrogenase (LPD) component of the PDH complex (E354K or H322Y). Such a mutation in lpd led to a PDH complex that was functional in an anaerobic culture as seen by restoration of anaerobic growth of a pflB, ldhA double mutant of E. coli utilizing a PDH- and alcohol dehydrogenase-dependent homoethanol fermentation pathway. The glutamate at position 354 in LPD was systematically changed to all of the other natural amino acids to evaluate the physiological consequences. These amino acid replacements did not affect the PDH-dependent aerobic growth. With the exception of E354M, all changes also restored PDH-dependent anaerobic growth of and fermentation by an ldhA, pflB double mutant. The PDH complex with an LPD alteration E354G, E354P or E354W had an approximately 20-fold increase in the apparent K(i) for NADH compared with the native complex. The apparent K(m) for pyruvate or NAD(+) for the mutated forms of PDH was not significantly different from that of the native enzyme. A structural model of LPD suggests that the amino acid at position 354 could influence movement of NADH from its binding site to the surface. These results indicate that glutamate at position 354 plays a structural role in establishing the NADH sensitivity of LPD and the PDH complex by restricting movement of the product/substrate NADH, although this amino acid is not directly associated with NAD(H) binding.
[Show abstract][Hide abstract] ABSTRACT: Bacillus coagulans is a ubiquitous soil bacterium that grows at 50-55 °C and pH 5.0 and ferments various sugars that constitute plant biomass to L (+)-lactic acid. The ability of this sporogenic lactic acid bacterium to grow at 50-55 °C and pH 5.0 makes this organism an attractive microbial biocatalyst for production of optically pure lactic acid at industrial scale not only from glucose derived from cellulose but also from xylose, a major constituent of hemicellulose. This bacterium is also considered as a potential probiotic. Complete genome sequence of a representative strain, B. coagulans strain 36D1, is presented and discussed.
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli KO11 (ATCC 55124) was engineered in 1990 to produce ethanol by chromosomal insertion of the Zymomonas mobilis pdc and adhB genes into E. coli W (ATCC 9637). KO11FL, our current laboratory version of KO11, and its parent E. coli W were sequenced, and contigs assembled into genomic sequences using optical NcoI restriction maps as templates. E. coli W contained plasmids pRK1 (102.5 kb) and pRK2 (5.4 kb), but KO11FL only contained pRK2. KO11FL optical maps made with AflII and with BamHI showed a tandem repeat region, consisting of at least 20 copies of a 10-kb unit. The repeat region was located at the insertion site for the pdc, adhB, and chloramphenicol-resistance genes. Sequence coverage of these genes was about 25-fold higher than average, consistent with amplification of the foreign genes that were inserted as circularized DNA. Selection for higher levels of chloramphenicol resistance originally produced strains with higher pdc and adhB expression, and hence improved fermentation performance, by increasing the gene copy number. Sequence data for an earlier version of KO11, ATCC 55124, indicated that multiple copies of pdc adhB were present. Comparison of the W and KO11FL genomes showed large inversions and deletions in KO11FL, mostly enabled by IS10, which is absent from W but present at 30 sites in KO11FL. The early KO11 strain ATCC 55124 had no rearrangements, contained only one IS10, and lacked most accumulated single nucleotide polymorphisms (SNPs) present in KO11FL. Despite rearrangements and SNPs in KO11FL, fermentation performance was equal to that of ATCC 55124.
[Show abstract][Hide abstract] ABSTRACT: Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.
Proceedings of the National Academy of Sciences 11/2011; 108(47):18920-5. DOI:10.1073/pnas.1111085108 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microaeration (injecting air into the headspace) improved the fermentation of hemicellulose hydrolysates obtained from the phosphoric acid pretreatment of sugarcane bagasse at 170°C for 10 min. In addition, with 10% slurries of phosphoric acid pretreated bagasse (180°C, 10 min), air injection into the headspace promoted xylose utilization and increased ethanol yields from 0.16 to 0.20 g ethanol/g bagasse dry weight using a liquefaction plus simultaneous saccharification and co-fermentation process (L+SScF). This process was scaled up to 80 L using slurries of acid pretreated bagasse (96 h incubation; 0.6L of air/min into the headspace) with ethanol yields of 312-347 L (82-92 gal) per tone (dry matter), corresponding to 0.25 and 0.27 g/g bagasse (dry weight). Injection of small amounts of air into the headspace may provide a convenient alternative to subsurface sparging that avoids problems of foaming, sparger hygiene, flotation of particulates, and phase separation.
[Show abstract][Hide abstract] ABSTRACT: Furfural is an important fermentation inhibitor in hemicellulose sugar syrups derived from woody biomass. The metabolism of
furfural by NADPH-dependent oxidoreductases, such as YqhD (low Km for NADPH), is proposed to inhibit the growth and fermentation of xylose in Escherichia coli by competing with biosynthesis for NADPH. The discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce
furfural provided a new approach to improve furfural tolerance. Strains that produced ethanol or lactate efficiently as primary
products from xylose were developed. These strains included chromosomal mutations in yqhD expression that permitted the fermentation of xylose broths containing up to 10 mM furfural. Expression of fucO from plasmids was shown to increase furfural tolerance by 50% and to permit the fermentation of 15 mM furfural. Product yields
with 15 mM furfural were equivalent to those of control strains without added furfural (85% to 90% of the theoretical maximum).
These two defined genetic traits can be readily transferred to enteric biocatalysts designed to produce other products. A
similar strategy that minimizes the depletion of NADPH pools by native detoxification enzymes may be generally useful for
other inhibitory compounds in lignocellulosic sugar streams and with other organisms.
[Show abstract][Hide abstract] ABSTRACT: The high fermentation cost of lactic acid is a barrier for polylactic acid (PLA) to compete with the petrochemical derived plastics. In order to lower the cost of lactic acid, the industry needs a microorganism that can ferment various sugars at high temperature (50°C) and at the same time using low cost mineral salts (MS) medium. One such bacterium, BL1, was isolated at 50°C and identified as Bacillus licheniformis. BL1 can ferment glucose to optically pure l-lactate with a maximum specific productivity of 7.8 g/hl in LB medium and 0.7 g/hl in MS medium at 50°C. BL1 can also consume 10% and 15% glucose in 20 and 48 h, respectively. After serial transfer of BL1 and BL2 in different concentrations of xylose and MS medium respectively, the final mutant BL3 could efficiently ferment glucose and xylose with specific productivity of 1.9 g/hl and 1.2g/hl in strict MS medium.
[Show abstract][Hide abstract] ABSTRACT: Lactic acid is used as an additive in foods, pharmaceuticals, and cosmetics, and is also an industrial chemical. Optically pure lactic acid is increasingly used as a renewable bio-based product to replace petroleum-based plastics. However, current production of lactic acid depends on carbohydrate feedstocks that have alternate uses as foods. The use of non-food feedstocks by current commercial biocatalysts is limited by inefficient pathways for pentose utilization. B. coagulans strain 36D1 is a thermotolerant bacterium that can grow and efficiently ferment pentoses using the pentose-phosphate pathway and all other sugar constituents of lignocellulosic biomass at 50°C and pH 5.0, conditions that also favor simultaneous enzymatic saccharification and fermentation (SSF) of cellulose. Using this bacterial biocatalyst, high levels (150-180 g l(-1)) of lactic acid were produced from xylose and glucose with minimal by-products in mineral salts medium. In a fed-batch SSF of crystalline cellulose with fungal enzymes and B. coagulans, lactic acid titer was 80 g l(-1) and the yield was close to 80%. These results demonstrate that B. coagulans can effectively ferment non-food carbohydrates from lignocellulose to L: (+)-lactic acid at sufficient concentrations for commercial application. The high temperature fermentation of pentoses and hexoses to lactic acid by B. coagulans has these additional advantages: reduction in cellulase loading in SSF of cellulose with a decrease in enzyme cost in the process and a reduction in contamination of large-scale fermentations.
[Show abstract][Hide abstract] ABSTRACT: Previous results have demonstrated that the silencing of adjacent genes encoding NADPH-dependent furfural oxidoreductases (yqhD dkgA) is responsible for increased furfural tolerance in an E. coli strain EMFR9 [Miller et al., Appl Environ Microbiol 75:4315-4323, 2009]. This gene silencing is now reported to result from the spontaneous insertion of an IS10 into the coding region of yqhC, an upstream gene. YqhC shares homology with transcriptional regulators belonging to the AraC/XylS family and was shown to act as a positive regulator of the adjacent operon encoding YqhD and DkgA. Regulation was demonstrated by constructing a chromosomal deletion of yqhC, a firefly luciferase reporter plasmid for yqhC, and by a direct comparison of furfural resistance and NADPH-dependent furfural reductase activity. Closely related bacteria contain yqhC, yqhD, and dkgA orthologs in the same arrangement as in E. coli LY180. Orthologs of yqhC are also present in more distantly related Gram-negative bacteria. Disruption of yqhC offers a useful approach to increase furfural tolerance in bacteria.
[Show abstract][Hide abstract] ABSTRACT: Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50-55 °C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55 °C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35 °C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55 °C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production.
[Show abstract][Hide abstract] ABSTRACT: Hexose and pentose sugars from phosphoric acid pretreated sugarcane bagasse were co-fermented to ethanol in a single vessel (SScF), eliminating process steps for solid-liquid separation and sugar cleanup. An initial liquefaction step (L) with cellulase was included to improve mixing and saccharification (L+SScF), analogous to a corn ethanol process. Fermentation was enabled by the development of a hydrolysate-resistant mutant of Escherichia coli LY180, designated MM160. Strain MM160 was more resistant than the parent to inhibitors (furfural, 5-hydroxymethylfurfural, and acetate) formed during pretreatment. Bagasse slurries containing 10% and 14% dry weight (fiber plus solubles) were tested using pretreatment temperatures of 160-190°C (1% phosphoric acid, 10 min). Enzymatic saccharification and inhibitor production both increased with pretreatment temperature. The highest titer (30 g/L ethanol) and yield (0.21 g ethanol/g bagasse dry weight) were obtained after incubation for 122 h using 14% dry weight slurries of pretreated bagasse (180°C).
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli strains (KJ060 and KJ073) that were previously developed for succinate production have now been modified for malate production.
Many unexpected changes were observed during this investigation. The initial strategy of deleting fumarase isoenzymes was
ineffective, and succinate continued to accumulate. Surprisingly, a mutation in fumarate reductase alone was sufficient to
redirect carbon flow into malate even in the presence of fumarase. Further deletions were needed to inactivate malic enzymes
(typically gluconeogenic) and prevent conversion to pyruvate. However, deletion of these genes (sfcA and maeB) resulted in the unexpected accumulation of d-lactate despite the prior deletion of mgsA and ldhA and the absence of apparent lactate dehydrogenase activity. Although the metabolic source of this d-lactate was not identified, lactate accumulation was increased by supplementation with pyruvate and decreased by the deletion
of either pyruvate kinase gene (pykA or pykF) to reduce the supply of pyruvate. Many of the gene deletions adversely affected growth and cell yield in minimal medium
under anaerobic conditions, and volumetric rates of malate production remained low. The final strain (XZ658) produced 163
mM malate, with a yield of 1.0 mol (mol glucose−1), half of the theoretical maximum. Using a two-stage process (aerobic cell growth and anaerobic malate production), this
engineered strain produced 253 mM malate (34 g liter−1) within 72 h, with a higher yield (1.42 mol mol−1) and productivity (0.47 g liter−1 h−1). This malate yield and productivity are equal to or better than those of other known biocatalysts.
[Show abstract][Hide abstract] ABSTRACT: Consolidation of bioprocessing steps with lignocellulose is limited by hydrolysate toxicity, the fibrous nature of suspensions, and low activity of cellulase enzymes. Combinations of enzyme dose and treatment conditions improved the flow properties and pumping of acid-pretreated sugarcane bagasse slurries (10% dry weight). Low levels of cellulase enzyme (0.1 and 0.5 FPU/g dry weight acid-pretreated bagasse) were found to reduce viscosities by 77-95% after 6 h, solubilizing 3.5% of the bagasse dry weight. Flow of slurries through small funnels was a useful predictor of success with centrifugal and diaphragm pumps. Equations were derived that describe viscosity and solubilized carbohydrates as a function of time and cellulase dosage. Blending of acid-pretreated bagasse (10% dry weight) with suspensions of acid-pretreated bagasse (10% dry weight) that had been previously digested with cellulase enzymes (low viscosity) did not increase viscosity in a linear fashion. Viscosity of these mixtures remained relatively constant until a threshold level of new fiber was reached, followed by a rapid increase with further additions. Up to 35% fresh acid-pretreated bagasse could be blended with enzyme-digested fiber (5.0 FPU/g dry weight acid-pretreated fiber; 6 h) with only a modest increase in viscosity. The smooth surfaces of enzyme-treated fiber are proposed to hinder the frequency and extent of interactions between fibrils of fresh fiber particles (acid-pretreated) until a threshold concentration is achieved, after which fiber interactions and viscosity increase dramatically. These results were used to model the viscosity in an ideal continuous stirred tank reactor (liquefaction) as a function of residence time and enzyme dosage.
[Show abstract][Hide abstract] ABSTRACT: Production of fuels and chemicals through microbial fermentation of plant material is a desirable alternative to petrochemical-based production. Fermentative production of biorenewable fuels and chemicals requires the engineering of biocatalysts that can quickly and efficiently convert sugars to target products at a cost that is competitive with existing petrochemical-based processes. It is also important that biocatalysts be robust to extreme fermentation conditions, biomass-derived inhibitors, and their target products. Traditional metabolic engineering has made great advances in this area, but synthetic biology has contributed and will continue to contribute to this field, particularly with next-generation biofuels. This work reviews the use of metabolic engineering and synthetic biology in biocatalyst engineering for biorenewable fuels and chemicals production, such as ethanol, butanol, acetate, lactate, succinate, alanine, and xylitol. We also examine the existing challenges in this area and discuss strategies for improving biocatalyst tolerance to chemical inhibitors.
BioMed Research International 04/2010; 2010(1110-7243):761042. DOI:10.1155/2010/761042 · 2.71 Impact Factor